Retinal Glial AQP4Internalization And Lysosomal Degradation Induced By Acute Ocular Hypertension And Its Molecular Mechnism

Aquaporin-4(AQP4), a membrane-bound water channel, targetsspecific membrane domains of retinal glial cells (e.g., retinal astrocytes,Müller cells), and enables the influx and efflux of water along hydrostaticand osmotic gradients. AQP4plays an important homeostatic role inmaintaining water balance, ionic/osmotic balance and intraocular pressure(IOP) in the healthy retina, as well as regulating water movement in thepathological edematous retina. The alteration of AQP4expression hasbeen observed in the ischemic-reperfused rat retina； Further studiesreported the localization of AQP4is also altered underischemia-reperfusion conditions and in light-injury. Thus, aberrations inretinal AQP4occur under ischemia-reperfusion conditions; however, themechanisms underlying these changes remain unclear.It is known that the endocytotic internalization of membrane-boundproteins (such as AQP4) can alter the subcellular localization of these proteins; moreover, this internalization may or may not be accompaniedby subsequent lysosomal sorting and degradation. Whether AQP4isinternalized in the ischemic-reperfused retina remains on open question.Accordingly, this research works including in vivo and in vitro studies arecarried out and aim to determine whether AQP4is internalized and/orlysosomally degraded in the rat retina during acute ocular hypertension(AOH) and reperfusion injury induced by ocular hypertension andexplore the mechanism underlining these processes. The results from thisstudy will clarify the molecular mediators of retinal edema induced byacute ocular hypertension in depth and supply an experimental basis forthe development of relevant therapeutic drugs for the treatment of retinaledema.Objective1. To investigate the spatial and temporal rules of AQP4（Aquaporin4）internalization as well as the subsequent lysosomal sorting anddegradation in the retinal injury induced by AOH and reperfusion, so asto disscuss the possible role of AQP4internalization and in the glialedema of retinae.2. To study the effects on AQP4internalization and lysosomal sortinginduced by hydrostatic pressure through the pressure-treated retinal glialcells and AQP4-transfected CHO (Chinese hamster ovary) cells, so as to explore the mechanisms of retinal AQP4internalization and lysosomalsorting under the AOH condition.Materials and Methods1.180Adult female Sprague–Dawley rats were used as animal modelsand randomly divided into several groups based on time intervals of acuteocular hypertention and time intervals of reperfusion after60min ofAOH. The rats were anesthetized with chloralhydrate and placed in astereotaxic frame. The anterior chamber of the left eye was cannulatedwith a26-gauge needle connected to a reservoir containing D-Hanksbuffer and the IOP was increased to110mm Hg. Five AOH groups wereestablished by maintaining an elevated IOP for5,10,30,60and90min.The six reperfusion groups each received a different duration ofreperfusion (0,1,2,4,8,12h) following60min of ischemia. Half of theright eyes in each group acted as controls. To control for the manipulationof rat eyes on AQP4expression, half of the right eyes were cannulatedwithout increasing the IOP as sham controls; the other half of the righteyes were not cannulated as normal control. At each pre-determined timepoint, eyes from part of the rats were enucleated and the wet-dryweighing method was applied to measure retina water content (RWC) ofrat retina in each group；Other part of rats were anesthetized withintraperitoneal administration of chloral hydrate and perfusedtranscardially through the ascending aorta, with200ml phosphate buffered saline (PBS) followed by250ml of solution of4%formaldehyde in0.1M PBS (pH7.4). The eyes were enucleated,bisected equatorially, and the posterior halves were immersed in fixative(4%paraformaldehyde) for2hr. Portions of the fixed posterior halveswere infiltrated with increasing concentrations of sucrose, followed byembedding in optimum-cutting temperature (OCT) medium, and then the12-μm sections were sliced by a cryo-cut cryostat microtome (Leica,Germany). The remaining portions were processed as wholemounts, inwhich the retinae were dissected, flattened by four (4) radial cuts, fixedfor an additional30min and mounted vitreal side-up on gelatine-coatedslides. Hematoxylin and eosin staining (HE staining) was used to observethe morphological changes of rat retina in each group；double-immunofluorescence labelings(sections) were applied to identitythe retinal AQP4+cells and detect the co-expression of AQP4and earlyendosome marker EEA1（Early Endosome Antigen1），AQP4and lateendosome marker MPR (mannose-6-phosphate receptor), AQP4andlysosome marker LAMP1(lysosomal-associated membrane protein-1)，and the numbers of single-labeled AQP4+cells and double-labeledAQP4+/EEA1+and AQP4+/LAMP1+cells were estimated in the retina,the ratio of double-labeled AQP4+/EEA1+and AQP4+/LAMP1+cells tosingle-labeled AQP4+cells was then calculated.2. Primary rat retinal glial cells were prepared from newborn pups (postnatal day15) and pretreated by20nM laminin protein. Theexpression of AQP4in primary rat retinal glial cells andAQP4-transfected CHO cells were detected by immunofluorescentstainings. Both of the cells were randomly divided into control groupsand pressure-treated groups. The control groups received no treatment.The pressure-treated groups were exposed to0.02MPa mechanicalpressure for3h. Double-immunofluorescence labelings were applied todetect the co-expression of AQP4and early endosome marker EEA1（Early Endosome Antigen1），AQP4and late endosome marker MPR(mannose-6-phosphate receptor), AQP4and lysosome markerLAMP1(lysosomal-associated membrane protein-1) in each kind of thecells. The calcein-quenching method was employed to detect the changesof osmotic water permeability.Results1. In vivo studies: Comparing to the control, the RWC was increasedsignificantly in every AOH groups except the5min of AOH groups；Inthe reperfusion groups, the RWC reach its peak (92.76%, P＜0.05) at1h,and then decreased to the minimum (86.33%, P＜0.05) at4h, followedby increasing with the duration of reperfusion8and12h. HE stainingshowed the retinal structure in experimental group was disorder arrangedwith marked edema. Double immunofluorescence labelings demonstratedthat AQP4+/GFAP+co-expressing astrocytes are located in the nerve fiber layer and ganglion cell layer, and bodies of AQP4+/GS+Müllercells in the inner nuclear layer；AQP4+/EEA1+co-expressing cells can beobserved at5,10,30,60,90min of AOH and all reperfusion groups. At60min of AOH, AQP4and EEA1coexpression climaxed, occupying72.97%±9.89%(P＜0.05, compared with adjacent time points). AQP4and MPR were co-localized from10to60min of AOH and0to8h ofreperfusion in rat retinae. The AQP4+/LAMP1+co-expressing cells canbe observed at1-12h of reperfusion, with co-expression increasingfollowed by a gradual decreas； At the4h reperfusion mark, AQP4andLAMP1coexpression climaxed, occupying55.60±13.03%(P<0.05,compared with adjacent time points).2. In vitro studies: Primary rat retinal glial cells were pretreated by20nM laminin protein, and AQP4clusters can be observed in themembrane. In AQP4-transfected CHO cells, AQP4was expressed in aplasma membrane pattern；In the pressure-treated groups of retinal glialand CHO cells, a large proportion of AQP4immunoreactivity hadtranslocated from membrane to cytoplasm. Double-immunofluorescencelabelings show AQP4in the cytoplasm co-expressed with EEA1(glialcells and CHO cells)，MPR(glial cells) and LAMP1(glial cells and CHOcells). Comparing to the control, calcein-quenching method show osmoticwater permeability of the pressure-treated glial cells and CHO cellsdecreased49.02%and58.81%，respectively(P＜0.05). Conclusions1. The injury of AOH and reperfusion can cause the edema anddisorder arranged of retina. During the course, the membranous AQP4can internalize into early endosome and late endosome, and the lysosomeis involved in degrading the internalized aquaporin-4during thereperfusion phase. The internalization of AQP4and its lysosomaldegradation played important roles during the development of retinaledema, which may serve as valuable therapeutic targets for managingAOH-reperfusional retinal injury.2. The membranous AQP4can internalize into endosome under themechnical pressure-treated condition, and the lysosome is involved indegrading the internalized aquaporin-4. Internalization/lysosomaldegradation of AQP4can lead to the decrease of osmotic waterpermeability, which maybe the adaptive reaction to the injury of AOH.